Fig 2: Inhibition of HER2 regulates PD-L1 expression by suppressing HER2/AKT/MTOR signaling in gastric cancer-derived organoids. The expression of the HER2/AKT/MTOR signaling pathway components were determined by (a) Western blot analysis. (b) Violin plots representing densitometric values of protein bands measured from 8a. Brightfield images and the calculated organoid area in cultures treated with (c) vehicle (control), or (d) Everolimus. n = 15 ROI from 3 different experiments, * p < 0.05 in comparison to vehicle. Immunofluorescences staining using antibodies specific for HER2 (green) and PD-L1 (red) using (e) vehicle (control), or (f) Everolimus treated organoid cultures. (g) Quantification of fluorescence intensity for PD-L1 expression in the control or Everolimus treated cultures. (h) Proposed mechanisms of anti-PD1 inhibition and suppression of MDSCs in a combination immunotherapy strategy for gastric cancer patients. Within the gastric TME, PMN-MDSCs override the checkpoint inhibition by releasing Arg1, iNOS and ROS. Pharmacological inhibition of ERBB2/HER2 could diminish PD-L1 +ve cells from the TME, improving patient responses to immunotherapy. Scale bar = 100 µm.

Fig 3: PD-L1 and HER2 expression in intestinal, diffuse and signet ring gastric cancer subtypes. H&E stain of (a,b) intestinal, (c,d) diffuse, and (e,f) signet ring gastric cancer subtypes. A higher magnification is demonstrated in panels (b), (d), and (f). (g) Quantification of HER2 and PD-L1 in the TMA using NanoString DSP. ns = not significant. Scale bar = 100 µm.

Fig 4: Immunohistochemistry and DSP analysis of FFPE tissue sections obtained from normal stomach and gastric tumor tissues. Immunohistochemical staining of representative ROIs for (a) HER2 and (b) PD-L1. DSP analysis of representative ROIs selected from (c) gastric cancer tissues showing staining for PanCK, SMA and CD68. DSP analysis of the whole tissue array (d) and a higher magnification of the selected tissue with ROI selection for Digital Spatial profiling and UV crosslinking. Analysis of the (e) tumor marker (PanCK) and immune marker (CD68) among PanCK-positive (+ve) and PanCK-negative (-ve) (CD68 + ve) areas of the FFPE tissues. (f) Quantitative expression of PD-L1 in HER2 -ve and HER2 +ve gastric cancer tissues. * p < 0.0001 compared to HER2 -ve gastric cancer tissue, n = 26 HER2 -ve and n = 46 HER2 +ve) (CD68 + ve) areas of the FFPE tissues. (f) Quantitative expression of PD-L1 in HER2 -ve and HER2 +ve gastric cancer tissues. * p < 0.0001 compared to HER2-ve gastric cancer tissue, n = 26 HER2 -ve and n = 46 HER2 +ve gastric cancer patients. Scale bar = 500 µm.

Fig 5: HER2 expression is significantly correlated with PD-L1 expression in gastric cancer tissues. Brightfield images showing morphological differences among huTGOs derived from intestinal (a,e) and diffuse (i,m) subtypes of gastric cancer tissues. Immunofluorescence staining of these organoids showed a high expression of (b–d,j–l) HER2 (red) and PD-L1 (green) in two of the organoid lines while others showed a negative expression (f–h,n–p) for both. Western blot (v) analysis of gastric cancer tissues showed a similar expression pattern. (q) shRNA knockdown of HER2 showed a downregulation of PD-L1 expression which was confirmed by (u) Western blot analysis and quantification. (r,s) Quantification of HER2 and PD-L1 positive cells showed a significant reduction in the expression of both genes after HER2 shRNA knockdown; * p < 0.0001. (t) Linear regression analysis showing a positive correlation of HER2 and PD-L1; Immunohistochemistry of PD-L1 (w) and HER2 (x) retained high expression levels among gastric cancer tissues, tissue-derived organoids and orthotopically transplanted organoids. Scale bar = 100 µm and 50 µm.